(1) Field of the Invention
This invention relates to a novel plasmid, a method for construction of the plasmid, a novel microorganism carrying the plasmid, and a method for cultivation of the microorganism and relates more particularly to a novel plasmid with DNA coding for extracellular production of such high-molecular substances as penicillinase, a novel microorganism transformed with the plasmid, and a method for extracellular production of such high-molecular substances as penicillinase by culturing the microorganism.
(2) Description of the Prior Art
Plasmid is a non-chromosomal gene of cyclic DNA found in a microorganism cell. Plasmid is currently being used as a means for recombination of microorganism gene and it is becoming more and more important in the field of fermentation industry.
Studies have recently been done on plasmids carrying foreign DNA having genetic information of metabolic products or specific demand for growth of microorganism, as shown in production of amino acids or peptides. Some plasmids have been introduced into host microorganisms to obtain transformants. Methods have been proposed for producing relatively low molecular compounds such as amino acids and peptides by culturing the transformants. However, the degree of propagation of plasmids carrying genes for production of high-molecular substances depends on the nature of host microorganisms and those plasmids have not effectively been expressed. Furthermore, no effective methods for cultivation of such transformants have been established.
It has not been possible to selectively obtain a certain extracellular high-molecular product by culturing a microorganism transformed with a plasmid carrying foreign DNA fragment having genetic information of extracellular production of high-molecular substances which are metabolic products of another microorganisms. Such extracellular production has not successfully been done even when a transformant of Escherichia species, which is usually used as a host microorganism, is used.
USP 4,411,994 of W. Gilbert et al discloses a process for producing specific proteins in bacteria and having them excreted from the bacterial cell. This process comprises inserting the DNA representing the desired non-bacterial protein or part of a protein by recombinant techniques into a plasmid or phage gene for either a periplasmic or an extracellular protein, hereinafter called a "carrier protein", transforming a bacterial host with the recombinant gene, and culturing the transformed host to excrete the protein. The process of this patent provides a means for producing a selected protein by employing a gene for a carrier protein which has a leader sequence of hydrophobic amino acids at its amino terminus.
Cell wall of Escherichia coli which has often been used for production of useful physiologically active substances, consists of three kinds of membrane: inner membrane, peptide glycan and outer membrane. The space between the inner and outer membrane is called the periplasmic space. The process of USP4,411,994 has succeeded in the excretion of the products within the periplasmic space but not within the culture medium through the outer membrane or outside the bacterial host cell. In the present invention, by inserting the DNA having genetic information of extracellular production of high-molecular substances into the plasmid to obtain a hybrid plasmid and culturing the host transformed with the hybrid plasmid, it is possible to excrete useful physiologically active substances through the outer membrane and recover them directly from the culture medium. Thus, the present invention makes mass production of useful physiologically active substances possible, which has never been possible by the prior art process.